Tuberculosis (TB) is one of the most frightening health problems facing the world today and the outlook for the TB crisis is ominous as a result of the TB-HIV syndemic and increasing reports of multiply, extensively and even totally drug resistant (MDR, XDR, TDR) Mycobacterium tuberculosis (Mtb) strains. New drugs that target novel Mtb pathways are desperately needed. All bacteria possess a highly conserved Sec pathway that exports proteins from the cytoplasm to the bacterial cell envelope or host environment. Among the proteins exported by the Sec system are proteins with essential functions or roles in virulence, making the Sec pathway an attractive drug target. One of the central components of the Sec pathway is the SecA ATPase that provides energy from ATP hydrolysis to drive protein translocation across the membrane. There are no mammalian homologues of SecA, which makes it an appealing target. Like other bacteria, Mtb has a functional Sec pathway. In mycobacteria, the canonical SecA is named SecA1. The goals of the R21 phase of this two stage proposal are to definitively prove Mtb SecA1 is essential for Mtb growth and survival in vitro and during in vivo infection, using conditional Mtb secA1 mutants. Also as part of the R21 phase, we will develop whole-cell assays that can be used in quantitative high-throughput screening (qHTS) for identifying inhibitors of Mtb SecA1. In the R33 phase of the project, we will automate and validate the qHTS assays and undertake pilot screening to assess assay performance as measured by the ability to identify compounds that inhibit Mtb SecA1. We will additionally solve the structure of a small set of inhibitors bound to Mtb SecA1 as a way to help understand inhibitor mode of action. The proposed work will validate Mtb SecA1 as a new drug target and, because SecA is highly conserved among bacteria, the results may also aid future drug design directed at SecA targets of other drug-resistant bacterial pathogens.